Regulation of blood flow and metabolic rate are closely coupled in striated muscle, but the pathways that specifically enable vasoactive metabolites to produce integrated arteriolar responses remain elusive. We have identified endothelial cells (EC), and EC Ca 2+ changes, as essential for metabolic vasodilation in resistance arterioles. This dilation involves, KATP channels, nitric oxide and adenosine, but how KATP channel activity relates to the actions of NO and adenosine is not clear. Importantly, the contribution of NO to this dilation is most prominent in ischemic conditions, i.e. mechanisms of metabolic vasodilation are modified by flow. The studies will use cremaster muscle of anesthetised mice to determine the mechanisms of this response in free flow and ischemic conditions. Intravital confocal microscopy will be used to quantify EC Ca 2+ and arteriolar diameter changes during skeletal muscle contraction. We will test 3 hypotheses that are based on our preliminary findings. Hyp. I: In metabofic vasodilation, KATP channels are required for the production of EC Ca 2+ changes. Hyp. Il: EC Ca 2+ changes are essential for metabolic vasodilation in all flow conditions. These changes consist of two components - an early increase mobilised from intracellular stores, and an oscillatory component dependent on extracellular calcium sources. Hyp. IIl: (Part A) NO of endothelial origin contributes significantly to the metabolic vasodilation that occurs during ischemic conditions, but is a much less important component of the response during free flow. (Part B) NO from non-endothelially located nNOS also contributes to metabofic vasodilation (particularly during ischemia) both by a direct effect on SMCs and by stimulating release of NO from endothelial cells. The studies will contribute to the general understanding of how local responses to physiological signals are integrated in blood vessel walls, and will contribute significantly to the broad goal of understanding the metabolic response to exercise. A full understanding of the mechanisms underlying exercise is relevant to many areas of human health.